Nucleotide Excision Repair Factors XPC Research Articles

Structural and dynamical insights into the PH domain of p62 in human TFIIH.

TFIIH is a crucial transcription and DNA repair factor consisting of the seven-subunit core. The core subunit p62 contains a pleckstrin homology domain (PH-D), which is essential for locating TFIIH at transcription initiation and DNA damage sites, and two BSD (BTF2-like transcription factors, synapse-associated proteins and DOS2-like proteins) domains. A recent cryo-electron microscopy (cryo-EM) structure of human TFIIH visualized most parts of core, except for the PH-D. Here, by nuclear magnetic resonance spectroscopy we have established the solution structure of human p62 PH-D connected to the BSD1 domain by a highly flexible linker, suggesting the flexibility of PH-D in TFIIH. Based on this dynamic character, the PH-D was modeled in the cryo-EM structure to obtain the whole human TFIIH core structure, which indicates that the PH-D moves around the surface of core with a specific but limited spatial distribution; these dynamic structures were refined by molecular dynamics (MD) simulations. Furthermore, we built models, also refined by MD simulations, of TFIIH in complex with five p62-binding partners, including transcription factors TFIIEα, p53 and DP1, and nucleotide excision repair factors XPC and UVSSA. The models explain why the PH-D is crucially targeted by these factors, which use their intrinsically disordered acidic regions for TFIIH recruitment.

Protective role of histone deacetylase 4 from ultraviolet radiation-induced DNA lesions.

Ultraviolet B (UVB) exposure is a core factor that leads to skin disease or carcinogenesis through the insufficient repair of DNA lesions. UVB-induced DNA lesions are mainly removed by the nucleotide excision repair (NER) mechanism. The expression of histone deacetylase 4 (HDAC4) is altered in the skin upon UVB exposure, indicating its possible implication in UVB-induced DNA lesions repair. Here, we investigated the role of HDAC4 in the NER removal of the main classes of UVB-induced DNA lesions consisting of cyclobutane pyrimidine dimersand pyrimidine (6-4) pyrimidone photoproducts (6-4PPs). We found that UVB irradiation increased HDAC4 expression at both the mRNA and protein levels. HDAC4 interacted with NER factor XPC, which played an important role in effectively removing the UVB-induced DNA lesions. This study provides an understanding of the HDAC4 function in DNA repair, which will allow the development of efficient strategies to protect the skin from UVR-induced diseases.

Polynuclear ruthenium organometallic compounds induce DNA damage in human cells identified by the nucleotide excision repair factor XPC.

Ruthenium organometallic compounds represent an attractive avenue in developing alternatives to platinum-based chemotherapeutic agents. While evidence has been presented indicating ruthenium-based compounds interact with isolated DNA in vitro, it is unclear what effect these compounds exert in cells. Moreover, the antibiotic efficacy of polynuclear ruthenium organometallic compounds remains uncertain. In the present study, we report that exposure to polynuclear ruthenium organometallic compounds induces recruitment of damaged DNA sensing protein Xeroderma pigmentosum Group C into chromatin-immobilized foci. Additionally, we observed one of the tested polynuclear ruthenium organometallic compounds displayed increased cytotoxicity against human cells deficient in nucleotide excision repair (NER). Taken together, these results suggest that polynuclear ruthenium organometallic compounds induce DNA damage in cells, and that cellular resistance to these compounds may be influenced by the NER DNA repair phenotype of the cells.

Nucleotide Excision Repair Factor XPC Ameliorates Prognosis by Increasing the Susceptibility of Human Colorectal Cancer to Chemotherapy and Ionizing Radiation.

Nucleotide excision repair (NER) is a DNA damage repair mechanism in mammals, but the relationship between NER and human colorectal cancer (HRC) progression has not been clarified yet. In this study, the expression of the NER genes XPA, XPC, XPF, XPG, ERCC1, and XPD was measured in normal and cancerous human colorectal tissue. Among them, only the XPC gene expression was significantly increased in colorectal cancer tissue. To establish the role of XPC in colorectal cancer, small interference RNA (siRNA) targeting XPC was used to knockdown the expression of XPC in HRC cell lines. In addition, an expression vector plasmid containing the XPC cDNA was constructed and stably transfected into HRC cell lines to overexpress the XPC gene. Interestingly, MTT and apoptosis assay demonstrated that XPC gene overexpression significantly increased the susceptibility of HRC cell lines to cisplatin and X-ray radiation. In order to study the relationship between XPC expression and the progression of HRC, XPC expression was measured in 167 patients with colorectal cancer. The results showed that patients with high XPC expression had longer survival time. Cox regression analysis showed that high XPC expression might be a potential predictive factor for colorectal cancer. In conclusion, XPC plays a key role in the susceptibility of colorectal cancer to chemotherapy and ionizing radiation and is associated with a good patients' prognosis.

Abstract 4155: USP11 regulates UV-induced DNA damage repair and cell survival associated with skin cancer

Abstract UV radiation is a major risk factor for skin cancer, the most common cancer in the United States. UV exposure leads to DNA damage by formation of dimers between adjacent pyrimidine bases. The nucleotide excision repair (NER) process is responsible for removal of UV-induced DNA damage. When the damage is left unrepaired due to inefficient NER in disorders like xeroderma pigmentosum, it leads to skin tumorigenesis. Although the main players of the NER pathway have been characterized, the molecular regulation of the pathway is less understood. Understanding the regulation, particularly post-translational regulation, of the NER pathway has the potential to yield modulators of NER efficiency to prevent skin cancer. We found that chronic UV irradiation led to down-regulation of ubiquitin specific peptidase 11 (USP11) in mouse skin tissue. We also found that USP11 is decreased in skin tumors in mice and humans. Moreover, high-throughput proteomics analysis had predicted that USP11 may interact with NER factor XPC. Hence, we undertook this study to determine the role of USP11 in NER and cellular response to UV. We found that USP11 promoted the NER pathway by regulating XPC activity. USP11 promoted XPC deubiquitination and retention at the DNA damage sites. We also found that USP11 interaction with ubiquitinated XPC was enhanced post-UV irradiation, and that USP11 was recruited to the chromatin post-UV damage. Further, squamous cell carcinoma cells showed reduced survival with inhibition of USP11 after UV exposure. Our findings indicate that USP11 plays an important role in maintaining NER capacity, and suggest that USP11 acts as a tumor suppressor via its role in DNA repair. Citation Format: Palak Shah, Lei Qiang, Seungwon Yang, Keyoumars Soltani, Yu-Ying He. USP11 regulates UV-induced DNA damage repair and cell survival associated with skin cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4155.

Dataset for the NMR structure of the intrinsically disordered acidic region of XPC bound to the PH domain of TFIIH p62.

The global genome nucleotide excision repair factor XPC firstly detects DNA lesions and then recruits a ten-subunit complex TFIIH through binding to the subunit p62 to unwind the damaged DNA for excision repair. This data article contains detailed nuclear magnetic resonance (NMR) restraints (nuclear Overhauser enhancement (NOE)-derived distance restraints, dihedral angle restraints, and hydrogen bond restraints) used for the structure determination of the complex formed between the intrinsically disordered acidic region of XPC and the pleckstrin homology (PH) domain of TFIIH p62, related to the recent work entitled “Structural insight into the mechanism of TFIIH recognition by the acidic string of the nucleotide excision repair factor XPC.” [1].

Tripartite DNA Lesion Recognition and Verification by XPC, TFIIH, and XPA in Nucleotide Excision Repair

Transcription factor IIH (TFIIH) is essential for both transcription and nucleotide excision repair (NER). DNA lesions are initially detected by NER factors XPC and XPE or stalled RNA polymerases, but only bulky lesions are preferentially repaired by NER. To elucidate substrate specificity in NER, we have prepared homogeneous human ten-subunit TFIIH and its seven-subunit core (Core7) without the CAK module and show that bulky lesions in DNA inhibit the ATPase and helicase activities of both XPB and XPD in Core7 to promote NER, whereas non-genuine NER substrates have no such effect. Moreover, the NER factor XPA activates unwinding of normal DNA by Core7, but inhibits the Core7 helicase activity inthe presence of bulky lesions. Finally, the CAK module inhibits DNA binding by TFIIH and thereby enhances XPC-dependent specific recruitment of TFIIH. Our results support a tripartite lesion verification mechanism involving XPC, TFIIH, and XPA for efficient NER.

Structural Insight into the Mechanism of TFIIH Recognition by the Acidic String of the Nucleotide Excision Repair Factor XPC

In global genome repair (GGR), XPC detects damaged nucleotides and recruits TFIIH complex. The small acidic region of XPC binds to the pleckstrin homology (PH) domain of TFIIH subunit p62; however, the recognition mechanism remains elusive. Here, we use nuclear magnetic resonance to present the tertiary structure of XPC bound to the PH domain. The XPC acidic region forms a long string stabilized by insertion of Trp133 and Val136 into two separate hollows of the PH domain, coupled with extensive electrostatic contacts. Analysis of several XPC mutants revealed that particularly Trp133 is essential for binding to the PH domain. In cell lines stably expressing mutant XPC, alanine substitution at Trp133 or Trp133/Val136 compromised UV resistance, recruitment of TFIIH to DNA damage, and removal of UV-induced photoproducts from genomic DNA. These findings show how TFIIH complex is recruited by XPC to damaged DNA, advancing our understanding of the early stage of GGR.

Real-time and simultaneous monitoring of the phosphorylation and enhanced interaction of p53 and XPC acidic domains with the TFIIH p62 subunit.

Posttranslational modifications have critical roles in diverse biological processes through interactions. Tumor-suppressor protein p53 and nucleotide excision repair factor XPC each contain an acidic region, termed the acidic transactivation domain (TAD) and acidic fragment (AF), respectively, that binds to the pleckstrin homology (PH) domain of the p62 subunit of the transcription factor TFIIH. Human p53-TAD contains seven serine and two threonine residues, all of which can be phosphorylated. Similarly, XPC-AF contains six serine and two threonine residues, of which Thr117, Ser122 and Ser129 have been reported as phosphorylation sites in vivo, although their phosphorylation roles are unknown. Phosphorylation of Ser46 and Thr55 of p53-TAD increases its binding ability; however, the role of XPC-AF phosphorylation remains elusive. Here we describe a system for real-time and simultaneous monitoring of the phosphorylation and p62-PH affinity of p53-TAD and XPC-AF using nuclear magnetic resonance (NMR) spectroscopy. Unexpectedly, among seven reported kinases that presumably phosphorylate Ser46 and/or Thr55 of p53-TAD, only two specific and high-efficiency enzymes were identified: JNK2α2 for Ser46 and GRK5 for Thr55. During interaction with p62-PH, four different affinity complexes resulting from various phosphorylation states of p53-TAD by the kinases were identified. The kinetics of the site-specific phosphorylation reaction of p53-TAD and its affinity for p62-PH were monitored in real-time using the NMR system. Isothermic calorimetry showed that phosphorylation of Ser129 of XPC-AF increases binding to p62-PH. Although CK2 was predicted to phosphorylate Ser122, Ser129 and Ser140 from its sequence context, it specifically and efficiently phosphorylated only Ser129. Simultaneous monitoring of the phosphorylation and augmentation in p62-PH binding identified a key residue of p62-PH for contacting phosphorylated Ser129. In summary, we have established an NMR system for real-time and simultaneous monitoring of site-specific phosphorylation and enhancement of affinity between phosphorylation domains and their target. The system is also applicable to other posttranslational modifications.

Repair of cisplatin-induced DNA interstrand crosslinks by a replication-independent pathway involving transcription-coupled repair and translesion synthesis

DNA interstrand crosslinks (ICLs) formed by antitumor agents, such as cisplatin or mitomycin C, are highly cytotoxic DNA lesions. Their repair is believed to be triggered primarily by the stalling of replication forks at ICLs in S-phase. There is, however, increasing evidence that ICL repair can also occur independently of replication. Using a reporter assay, we describe a pathway for the repair of cisplatin ICLs that depends on transcription-coupled nucleotide excision repair protein CSB, the general nucleotide excision repair factors XPA, XPF and XPG, but not the global genome nucleotide excision repair factor XPC. In this pathway, Rev1 and Polζ are involved in the error-free bypass of cisplatin ICLs. The requirement for CSB, Rev1 or Polζ is specific for the repair of ICLs, as the repair of cisplatin intrastrand crosslinks does not require these genes under identical conditions. We directly show that this pathway contributes to the removal of ICLs outside of S-phase. Finally, our studies reveal that defects in replication- and transcription-dependent pathways are additive in terms of cellular sensitivity to treatment with cisplatin or mitomycin C. We conclude that transcription- and replication-dependent pathways contribute to cellular survival following treatment with crosslinking agents.

Nucleotide Excision Repair Factor XPC Enhances DNA Damage–Induced Apoptosis by Downregulating the Antiapoptotic Short Isoform of Caspase-2

XPC protein is a critical DNA damage recognition factor in nucleotide excision repair for which genetic deficiency confers a predisposition to cancer. In this study, we show that XPC has a function that is independent of its canonical function in DNA repair, potentially altering the interpretation of how XPC deficiency leads to heightened cancer susceptibility. XPC enhances apoptosis induced by DNA damage in a p53 nullizygous background, acting downstream of mitochondrial permeabilization and upstream of caspase-9 activation in the DNA damage-induced apoptosis cascade. We found that deficiency in XPC upregulated production of the short isoform of caspase-2 (casp-2S). This upregulation occurred at both protein and mRNA levels through repression of the caspase-2 promoter by XPC protein. Targeted RNAi-mediated downregulation of casp-2S-enhanced UV-induced apoptosis as well as activation of caspase-9 and caspase-6 in XPC-deficient cells, but not in XPC-proficient cells. In addition, XPC overexpression in various p53-deficient cancer cells resistant to cisplatin improved their sensitivity to cisplatin-induced apoptosis. Given that casp-2S functions as an antiapoptotic protein, our findings suggest that XPC enhances DNA damage-induced apoptosis through inhibition of casp-2S transcription. Together, these findings offer a mechanistic foundation to overcome the resistance of highly prevalent p53-deficient tumors to cell death induced by DNA-damaging therapeutic agents, by targeting strategies that inhibit the expression or function of casp-2S.

Enhanced Nucleotide Excision Repair in Human Fibroblasts Pre‐exposed to Ionizing Radiation

Cellular protection against deleterious effects of DNA damaging agents requires an intricate network of defense mechanisms known as the DNA damage response (DDR). Ionizing radiation (IR) mediated activation of the DDR induces a transcriptional upregulation of genes that are also involved in nucleotide excision repair (NER). This suggests that pre-exposure to X-rays might stimulate NER in human cells. Here, we demonstrate in normal human fibroblasts that UV-induced NER is augmented by pre-exposure to IR and that this increased repair is accompanied by elevated mRNA and protein levels of the NER factors XPC and DDB2. Furthermore, when IR exposure precedes local UV irradiation, the presence of XPC and DDB2 at the sites of local UV damages is increased. This increase might be p53 dependent, but the mechanism of X-ray specific stabilization of p53 is unclear as both X-rays and UV stabilize p53.

Abstract 186: NER factor XPC enhances DNA damage-induced apoptosis independently of p53 function

Abstract Xeroderma Pigmentosum complementation group C (XPC) is a 940-aa protein engaged in the DNA damage recognition during nucleotide excision repair (NER). It was reported that one of the XPC polymorphisms, Lys939Gln, is associated to cancer predisposition, although this variant did not affect NER. Thus, another mechanism must exist to contribute to this cancer susceptibility. In addition, this XPC genetic polymorphism correlates with the failure of imatinib treatment in patients with chronic myeloid leukemia. We reasoned that XPC must be influencing other functions unrelated to DNA repair. Here, we demonstrated that XPC can enhance the apoptosis induced by various DNA damaging agents, including ionizing radiation (IR), ultraviolet light (UV), cisplatin, and etoposide. In addition, XPC-mediated apoptosis is independent of p53, as reflected by the findings that knock-down of XPC expression compromised the UV or IR-induced apoptosis in p53-deficient Li-Fraumeni Syndrome (LFS) MDAH041 and HCT116 cell lines, while having no influence on apoptosis in their p53-proficient counterparts. Also, in response to UV irradiation, XPC deficiency did not affect the mitochondrial cytochrome c release or had only slight effect on the activation of caspase-3. However, XPC deficiency compromised the UV-induced activation of caspase-9. Therefore, XPC seems to function downstream of mitochondrial membrane permeabilization (MMP) and upstream of caspase-9 activation during DNA damage-induced apoptosis. We further demonstrated that XPC deficiency impaired the UV-induced caspase-2 activation, as reflected by reduced cleavage of long isoform of caspase-2 (CASP-2L) in XP-C cells compared to XPC-restored XP-C cells following UV irradiation. Meanwhile, the short isoform of caspase-2 (CASP-2S) was found to be induced in XP-C cells at early times following UV irradiation, while it remained unchanged in XPC-restored XP-C cells. Given that caspase 2 gene generates both proapoptotic (CASP-2L) and antiapoptotic (CASP-2S) isoforms by alternative splicing, our data suggest that XPC enhances DNA damage-induced apoptosis by inhibiting the production of anti-apoptotic CASP-2S via regulation of the alternative splicing of caspase-2. (Supported by NIH grants CA93413, ES2388 and ES12991). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 186. doi:10.1158/1538-7445.AM2011-186

The p38 Mitogen-activated Protein Kinase Augments Nucleotide Excision Repair by Mediating DDB2 Degradation and Chromatin Relaxation

The p38 MAPK is a family of serine/threonine protein kinases that play important roles in cellular responses to external stress signals, e.g. UV irradiation. To assess the role of p38 MAPK pathway in nucleotide excision repair (NER), the most versatile DNA repair pathway, we determined the efficiency of NER in cells treated with p38 MAPK inhibitor SB203580 and found that p38 MAPK is required for the prompt repair of UV-induced DNA damage CPD. We further investigated the possible mechanism through which p38 MAPK regulates NER and found that p38 MAPK mediates UV-induced histone H3 acetylation and chromatin relaxation. Moreover, p38 MAPK also regulates UV-induced DDB2 ubiquitylation and degradation via phosphorylation of the target protein. Finally, our results showed that p38 MAPK is required for the recruitment of NER factors XPC and TFIIH to UV-induced DNA damage sites. We conclude that p38 MAPK regulates chromatin remodeling as well as DDB2 degradation for facilitating NER factor assembly.

Recruitment of the nucleotide excision repair endonuclease XPG to sites of UV-induced dna damage depends on functional TFIIH.

The structure-specific endonuclease XPG is an indispensable core protein of the nucleotide excision repair (NER) machinery. XPG cleaves the DNA strand at the 3' side of the DNA damage. XPG binding stabilizes the NER preincision complex and is essential for the 5' incision by the ERCC1/XPF endonuclease. We have studied the dynamic role of XPG in its different cellular functions in living cells. We have created mammalian cell lines that lack functional endogenous XPG and stably express enhanced green fluorescent protein (eGFP)-tagged XPG. Life cell imaging shows that in undamaged cells XPG-eGFP is uniformly distributed throughout the cell nucleus, diffuses freely, and is not stably associated with other nuclear proteins. XPG is recruited to UV-damaged DNA with a half-life of 200 s and is bound for 4 min in NER complexes. Recruitment requires functional TFIIH, although some TFIIH mutants allow slow XPG recruitment. Remarkably, binding of XPG to damaged DNA does not require the DDB2 protein, which is thought to enhance damage recognition by NER factor XPC. Together, our data present a comprehensive view of the in vivo behavior of a protein that is involved in a complex chromatin-associated process.

Tumor suppressor p53 dependent recruitment of nucleotide excision repair factors XPC and TFIIH to DNA damage

Functional tumor suppressor p53 is mainly required for efficient global genomic repair (GGR), a subpathway of nucleotide excisions repair (NER). In this study, the regulatory effect of p53, on the spaciotemporal recruitment of XPC and TFIIH to DNA damage sites, was investigated in repair-proficient and -deficient human cells in situ. Photoproducts were induced through micropore UV irradiation of discrete subnuclear areas of intact cells and the specific lesions, as well as recruited repair factors, were detected by immunofluorescent intensity and density of the damaged DNA subnuclear spots (SNS). Both cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts (6-4PP) were visualized in situ at SNS within irradiated nuclear foci. The in situ repair kinetics revealed that p53-WT normal fibroblasts are proficient for the repair of both CPD and 6-4PP, whereas, p53-Null Li-Fraumeni syndrome (LFS) fibroblasts fail to efficiently repair CPD but not 6-4PP. Colocalization experiments of the NER factors showed that in normal human cells, XPC and TFIIH are rapidly and efficiently recruited to DNA damage within SNS. By contrast, recruitment of both XPC and TFIIH to DNA damage in SNS occurred much less efficiently in p53-Null or p53-compromised cells. The total cellular levels of XPC and XPB were similar in both p53-WT and -Null cells and remained unchanged up to 24 h following UV irradiation. The results also showed that dispersal of recruited XPC and TFIIH from DNA damage SNS occurs within a short period after DNA damage. Such dispersal requires functional XPA, XPF and XPG proteins. Taken together, the results demonstrated that p53 plays a pronounced role in the damage recognition and subsequent assembly of repair machinery during GGR and the recruitment of XPC and TFIIH to CPD is p53-dependent. Most likely mechanism of this p53 action is through its downstream effector protein, DDB2.